Use of an Active Biological Substance in Abnormal Cellular and Viral Membrane Physiologies

ABSTRACT

An active biological substance is disclosed for use in abnormal cellular and viral membrane physiologies in human and mammal organisms. The active substance has diagnostic and/or therapeutic properties and contains or consists of at least one component selected from the group of substances including: histones, covalently modified histones, histone-like polypeptides, biologically active histone sequences and histone-like polypeptides as agents for stopping the supply to solid tumours over their blood vessels, for killing cells infected by virus and for killing tumour cells with disturbed lipid asymmetry.

This continuation application claims the benefit of priority pursuant to35 U.S.C. §120 to U.S. patent application Ser. No. 11/989,784, filedJul. 23, 2009, a 35 U.S.C. 371 national phase filing of InternationalPatent Application PCT/EP2006/007764, filed Aug. 4, 2006, which claimsthe benefit of priority to German Patent Application 102005037602.9,filed Aug. 5, 2005; each of which is hereby incorporated by reference inits entirety.

The invention relates to the use of an active biological substance formedical purposes in abnormal cellular and viral membrane physiologies.

Eukaryotic cell membranes are known to consist of proteins,carbohydrates and especially lipids, mainly cholesterol and alsophospholipids and glycosphingolipids.

The phospholipids are known to consist of sphingomyelin (SM) and of thephospholipids phosphatidylcholine (PC), phosphatidylethanolamine (PE)and phosphatidylserine (PS). Small amounts of other phospholipids alsoexist.

The ratios by quantity of the cell membrane building blocks differ inhumans and in various mammal species.

A cellular membrane consists of an outer layer and an inner layer.Cellular regulation processes ensure that the respective phospholipidcompositions are maintained in each layer. The phospholipid compositionsin the outer and inner layers are different. The phospholipids PE and PSoccur mainly in the inner layers, while the phospholipids PC and SMoccur in both layers of the cell membrane. These vertical lipidasymmetries in the outer and inner cell membrane layers are strictlyregulated in every eukaryotic cell. However, the cell membraneregulation in each cell is effected by different enzymes and transportproteins, which ensure that the predetermined proportions of theindividual lipid components in the outer and inner layers arepermanently maintained and that the vertical lipid asymmetrypredetermined for a healthy cell remains undisturbed.

Of particular note is the ATP-driven transport of the aminophospholipidsPE and PS by amino-phospholipid translocase (APLT) from the outer layersinto the inner layers of the cell membrane, such that, in healthy cells,it is possible to ensure permanently that the phospholipids PE and PSare maintained at the predetermined proportions in the inner layer.

This applies also to the endothelial cells of normal blood vessels, theouter layers of which delimit the inner face of the blood vessel walls.

If the milieu of healthy cells becomes pathologically altered, theregulation system of the healthy cells for maintaining the predeterminedlipid asymmetries in the bilayers of the cell membranes may becomeimpaired. The continuous ATP-driven transport of the phospholipids PEand PS from the outer layers into the inner layers of the cell membranesis no longer guaranteed if the healthy cells, e.g. healthy endothelialcells of blood vessels, are located in the vicinity of tumor cells. Thehigh growth rate of a solid tumor leads to an abnormal metabolicsituation in tumors, which situation is caused inter alia by oxygendeficiency and free oxygen radicals. Several factors lead todysregulation of the lipid asymmetry in the membranes of tumor cells.The dysregulation also carries over to the endothelial cells adjacent tothe tumor cells, as has been confirmed by experimental studies.

In addition to a pathologically disturbed cell milieu, which maypossibly lead to disturbance of the lipid symmetry in the layers of thecell membranes, such disturbance may also be caused by viruses, e.g.retroviruses, that have penetrated into cells.

A disturbed lipid symmetry in the layers of cell membranes is found whenanionic lipid constituents are present in considerable numbers in theouter layers of the cell membranes of cells, which are referred to belowas pathogenic and may have a malignant potential. The anionic lipidconstituents particularly involve phosphatidylserine (PS).

Abnormal cells of this kind, with pathologically disturbed verticallipid asymmetries in the cell membrane layers, are to be found inserious diseases. Among these are diseases involving solid tumors.

Pathological cells with disturbed lipid asymmetries in the bilayers oftheir cell membranes are often not detected in good time, because of thelack of early diagnosis, which is why surgical interventions followed bychemotherapy and/or radiotherapy are needed after a disease has becomemanifest, e.g. with solid tumors. After such an operation, the patientsare often left at risk of a renewed outbreak of the disease, since notall pathological cells have been able to be removed or killed off. Inthe patients affected, it is therefore possible that at some time cancercells will be able to develop again from the remaining pathogeneiccells, in which case the renewed outbreak of the disease is often nolonger treatable, since conventional chemotherapy and radiationprocedures of the kind used to treat the initial outbreaks of thedisease often fail when used to treat the renewed outbreak of thedisease, or they can no longer be tolerated by the patients because oftheir weakened condition.

A renewed outbreak of a disease, however, would be avoidable with anactive substance that can diagnose pathological cells and kill them offbefore they are able to manifest themselves outwardly as a cancerousdisease.

Experimental studies have also shown that eukaryotic cells infected byviruses have pathologically disturbed vertical lipid asymmetries in thecell membranes, e.g. in AIDS and in hepatitis C.

The viruses that infect cells also include enveloped viruses, in whichthe viral envelopes are derived from the membranes of their host cells.

Viruses can be classed in various virus families according to variousmain criteria (nature and shape of the genome, form of symmetry of thecapsids, and the presence of a membrane shell). The viruses withmembrane shells (viral envelopes) include the virus families below:

-   Flaviviridae-   Togaviridae-   Coronaviridae-   Arteriviridae-   Rhabdoviridae-   Paramyxoviridae-   Filoviridae-   Bornaviridae-   Orthomyxoviridae-   Bunyaviridae-   Arenaviridae-   Retroviridae-   Hepadnaviridae-   Herpesviridae-   Poxviridae-   Asfarviridae

In virus morphogenesis, after the replication processes of the envelopedviruses in their host cells, parts of the cellular membrane systems ofthe host cells are integrated into the viral envelopes of the viruses,such that the distrurbed lipid asymmetries of the cellular membraneparts are carried over into the viral envelopes. It is in this sensethat the expression “abnormal viral membrane physiology” is also to beunderstood here.

European patent 0149486 has disclosed the use of at least one histoneand/or at least one active histone section for diagnosis and forimmunotherapy, for treatment of endocrinological disturbances, and forcancer therapy.

European patent 0392315 has supplemented the above prior art bydisclosing that histone H1 in particular, or its active section, issuitable for cancer therapy.

European patent 0438756 and U.S. Pat. No. 5,780,432 have furtherdisclosed a medicament consisting of a cytostatic as a first activesubstance and of a histone, or its active section, as a second activesubstance, which active substances together exert a synergistic effectin cancer therapy and in the treatment of autoimmune diseases.

German patent application DE 197 15 149 A1 and the PCT application withthe international publication number WO 98/46252 disclose a therapeuticagent based on histone H1 or histone-like proteins or active partsthereof for the treatment of cancer cells, in particular of thehematogenic system, which have, as receptors, cell-membrane-resistantcore histones or core-histone-like polypeptides. The therapeutic histoneH1 active substance can also be a recombinant H1 subtype.

From J. J. Killian et al. in the journal Emerging Therapeutic Targets(1999) 3 (3), pages 454-468, “Cell Membrane Lipids as ExperimentalTherapeutic Targets” and from S. Ran et al. in the journal Clin. CancerDis. 2005 Feb. 15, 11 (4): 1551-62 “Antitumor Effects of a MonoclonalAntibody that binds Anionic Phospholipids on the Surface of Tumor BloodVessels in Mice”, it is known that anionic phospholipids, in particularphosphatidylserine, serve as targets for monoclonal antibodies inendothelial cells of tumor vessels in mice, with the aim of destroyingthe tumors by suppressing the supply of blood.

In European patent application 04011015.7 of May 7, 2004, it has alreadybeen proposed by us to use the active substance according to theinvention in particular for early diagnosis and/or preventive treatmentof virus-infected living cells, its efficacy being directed selectivelyagainst cell membranes, particularly phospholipid membranes ofvirus-infected cells, that are modified after the virus attack to act asan identifer of the virus attack, and also have histones in the cellmembranes which, in healthy cells not infected by viruses, have hithertoonly been identifiable in the nucleus.

From H. Zhao, K. J. Kinnunen et al. in the journal Biochemistry 2004,43; 10192-10202 “Interactions of Histone H1 with Phospholipids andComparison of its Binding to Giant Liposomes and Human LeukemicT-cells”, it is known that bovine histone H1 binds to phosphatidylserineof liposomes and leukemic T-cells and then destroys these.

The object of the invention is to make available a medical activesubstance which not only has an identifier function in respect ofdisease with solid tumors and/or in respect of viral disease in humansand mammals or in respect also of latent diseases of this kind, but atthe same time also has a cytostatic or virostatic action potential fordestroying pathogenic cells with disturbed lipid asymmetry, as ischaracteristic for cells infected by viruses and, in solid tumors, bothfor the tumor cells and also for the endothelial cells of the bloodvessels supplying the tumors.

The invention starts out from the recognition that the membranes ofpathogenic cells, in particular cells of solid tumors and/or cellsinfected by viruses, have a disturbed asymmetry of the lipid compositionin the membrane bilayers, characterized by increased presence of anionicphospholipids, in particular phosphatidylserine, in the outer layer ofthe membranes.

According to the invention, the object is achieved by an activebiological substance having diagnostic and/or therapeutic properties andcontaining or consisting of at least one component selected from thegroup of substances including: histones,] covalently modified histones,histone-like polypeptides, biologically active sequences of histones andhistone-like polypeptides, as agents for stopping supply to solid tumorsvia their blood vessels, for killing off cells infected by viruses, inabnormal cellular and/or viral membrane physiologies caused by defectiveregulation of the phospholipids in the outer cellular membrane areas.

Advantageous developments of the invention are set forth in thesubclaims and in the following description of advantageous examples ofuse.

The cellular membranes of solid tumors, but also the endothelial cellsof the blood vessels supplying the solid tumors especially to meet theirincreased oxygen demand, have, like the tumors too, a pathologicallydisturbed membrane physiology, attributable to defective regulation,suppression of anionic phospholipids, in particular phosphatidylserine,in the outer membrane layers.

The active substance according to the invention can therefore be used todamage the endothelial cells of blood vessels that supply the solidtumors. For example, recombinant human histone rH1.3 is used as activesubstance according to the invention. The invention is not limited tothis, and, accordingly, it is also possible for other Hi subtypes H1.0,H1.1, H1.2, H1.4, H1.6, H1.t and H1.x and their biologically activesections, but also the core histones, to be used according to theinvention as active substance.

Histone as active substance can be used in therapeutic doses of, forexample, 10⁻¹ to 10⁻² μM substantially without side effects on theorganism and its immune system.

Histone rH1.3 as a diagnostic and at the same time therapeutic substanceis conveyed through the blood stream into the blood vessels of tumors ina patient suffering from solid tumors. In the blood vessels, the activesubstance attacks the outsides of the membranes of the endothelial cellsthat form the inner walls of the pathological blood vessels in or on thesolid tumors.

As the histone (H1) used as active substance binds to phosphatidylserine(PS) and/or to membrane histones of the enothelial cells, thesubstantially unordered structure of H1 changes to an ordered structureH1α with α-helix components (amphipathic helix), which can beillustrated as follows

H1+nPS→H1α[PS]n

This results in an m-fold self-aggregation of H1a, which can be furthercharacterized by the relationship

mH1a[PS]n→{H1α[PS]n}m

The self-aggregations of the histones (here H1) on the membranes of theendothelial cells of the tumor blood vessels leads to apoptosis and topore formation in the endothelial cell membranes, which leads todestruction of the endothelial cells. This continuing process ofdestruction on a large number of endothelial cells finally leads tomassive vessel damage and therefore decreases the supply of oxygen andnutrients to the solid tumors.

The active substance according to the invention binds not only to themembranes of the endothelial cells of blood vessels on or in theinterior of tumors, but also binds to the membranes of virus-infectedcells which, on account of the virus attack, have a disturbed lipidasymmetry in their bilayers.

Finally, the active substance according to the invention opens up newdiagnostic and therapeutic possibilities in respect of cells that areinfected by viruses and that can be reached by the active substanceespecially, but not exclusively, via body fluids. The lysis of thevirus-infected cells, even before release of the viruses from their hostcells, means that these can be identified by the abnormal lipidasymmetries of the lipid double membranes of the host cells for theactive substance according to the invention, which, alone or incombination with cytostatics and/or virostatics, destroys thevirus-infected host cells and thus prevents replication of the viruses.

In many cases, a concentration of the active substance, e.g. rH1.3, of10⁻¹ to 10⁻² μM is sufficient.

Since the active substance according to the invention crosses theblood/brain barrier by itself and also as a vehicle for cytostatics andvirostatics, this also opens up new diagnostic or therapeuticpossibilities for treatment of diseases in the brain, particularly forearly diagnosis of brain cells that have an abnormal lipid asymmetry inthe lipid double membrane.

1. A method of destroying abnormal blood vessels, the method comprisingthe step of administering an active biological substance comprising H1histone to a mammal, wherein the abnormal blood vessels compriseendothelial cells having a disturbed lipid asymmetry in a membranebilayer; and wherein administration of the active biological substancedestroys the endothelial cells having a disturbed lipid asymmetry in amembrane bilayer, thereby destroying the abnormal blood vessels.
 2. Themethod according to claim 1, wherein the active biological substance isadministered in conjunction with a cytostatic, a virostatic, or both acytostatic and a virostatic.
 3. The method according to claim 1, whereinthe mammal is a human.
 4. The method according to claim 1, wherein thedisturbed lipid asymmetry includes an increased presence of anionicphospholipids in the membrane bilayer.
 5. The method according to claim4, wherein the anionic phospholipids include phosphatidylserine.
 6. Themethod according to claim 1, wherein the active biological substancecomprises about 1×10 ⁻¹ μM to about 1×10⁻² μM of H1histone.
 7. Themethod according to claim 1, wherein the H1 histone is H1.0, H1.1, H1.2,H1.3, H1.4, H1.6, H1.t, H1.x, or a biologically active section thereof.8. The method according to claim 1, wherein the abnormal blood vesselssupply a solid tumor.
 9. A method of diagnosing abnormal blood vessels,the method comprising the steps of: administering an active biologicalsubstance comprising H1 histone to a mammal suspected of having theabnormal blood vessels, wherein the abnormal blood vessels compriseendothelial cells having a disturbed lipid asymmetry in a membranebilayer, and wherein the active biological substance destroysendothelial cells having a disturbed lipid asymmetry in a membranebilayer; and detecting the presence of the destroyed endothelial cells,wherein the presence of the destroyed endothelial cells is indicative ofthe abnormal blood vessels.
 10. The method according to claim 9, whereinthe active biological substance is used in conjunction with a markermolecule for diagnostic purposes.
 11. The method according to claim 9,wherein the active biological substance is administered in conjunctionwith a cytostatic, a virostatic, or both a cytostatic and a virostatic.12. The method according to claim 9, wherein the mammal is a human. 13.The method according to claim 9, wherein the disturbed lipid asymmetryincludes an increased presence of anionic phospholipids in the outerlayer of the membrane.
 14. The method according to claim 13, wherein theanionic phospholipids include phosphatidylserine.
 15. The methodaccording to claim 9, wherein the active biological substance comprisesabout 1×10 ⁻¹ μM to about 1×10⁻² μM of H1 histone.
 16. The methodaccording to claim 9, wherein the H1 histone is H1.0, H1.1, H1.2, H1.3,H1.4, H1.6, H1.t, H1.x, or a biologically active section thereof.